AU711653B2

AU711653B2 - Inducible herbicide resistance

Info

Publication number: AU711653B2
Application number: AU66278/96A
Authority: AU
Inventors: Ian Jepson
Original assignee: Zeneca Ltd
Current assignee: Syngenta Ltd
Priority date: 1995-08-03
Filing date: 1996-08-02
Publication date: 1999-10-21
Anticipated expiration: 2016-08-02
Also published as: CN1197483A; BR9609886A; NO980450L; EP0843730A1; HUP9802867A2; BG102272A; AU6627896A; NO980450D0; HUP9802867A3; PL324821A1; NZ313750A; GB9515941D0; JPH11510695A; TR199800131T1; CZ29198A3; CA2224732A1; RU2181380C2; US6380463B1; WO1997006269A1; MX9800861A

Description

I WO 97/06269 PCT/GB96/01883

-I-

INDUCIBLE HERBICIDE RESISTANCE The present invention relates to DNA constructs and plants incorporating them. In particular it relates to promoter sequences for the expression of genes which confer herbicide resistance on plants.

Recent advances in plant biotechnology have resulted in the generation oftransgenic plants resistant to herbicide application. Herbicide tolerance has been achieved using a range of different transgenic strategies. One well documented example is the use the bacterial xenobiotic detoxifying gene phosphinothricin acetyl transferase (PAT) from Streptomyces hydroscopicus. Mutated genes of plant origin, for example the altered target site gene encoding acetolactate synthase (ALS) from Arabidopsis, have been successfully utilised to generate transgenic plants resistant to herbicide application. The PAT and ALS genes have been expressed under the control of strong constitutive promoter.

We propose a system where genes conferring herbicide tolerance would be expressed in an inducible manner dependent upon application of a specific activating chemical. This approach has a number of benefits for the farmer, including the following: 1. Inducible control of herbicide tolerance would alleviate any risk of yield penalties associated with high levels of constitutive expression of herbicide resistance genes.

This may be a particular problem as early stages of growth where high levels of transgene product may directly interfere with normal development. Alternatively high levels of expression of herbicide resistance genes may cause a metabolic drain for plant resources.

2. The expression of herbicide resistance genes in an inducible manner allows the herbicide in question to be used to control volunteers if the activating chemical is omitted during treatment.

3. The use of an inducible promoter to drive herbicide resistance genes will reduce the risk of resistant weed species becoming a major problem. If resistance genes were passed onto weed species from related crops, control could still be achieved with the herbicide in the absence of inducing chemical. This would particularly be relevant if the tolerance gene confirmed resistance to a total vegetative control herbicide which would be used (with no inducing chemical) prior to sowing the crop and potentially after the crop has been harvested. For example, it can be envisaged that herbicide WO 97/06269 PCT/GB96/01883 -2resistance in cereals, such as wheat, might outcross into the weed wild oats or that herbicide resistance in oil seed rape or canola could be transferred to wild brassicas thus conferring herbicide resistance to these already troublesome weeds. A further example is that the inducible expression of herbicide resistance in sugar beet will reduce the risk of wild sugar beet becoming a problem.

Several gene regulation systems (gene switches) are known and may be used for conferring inducible herbicide resistance on plants. Many such gene switches are described in the review by Gatz (Current Opinion in Biotechnology (1996) 7, 168-172) and include systems such as the tetracycline repressor gene switch, the Lac repressor system, copper inducible systems such as that based on ACE 1, salicylic acid inducible promoters including the PR-la system and systems based on sterioid hormones such as the glucocorticoid, progesterone and oestrogen receptor systems. Modifications of the glucocorticoid receptor systems which include the GAL 4 binding domain from yeast and the VP 16 activator are described by Aoyama et al, The Plant Cell, (1995) 7, 1773-1785 and it is envisaged that similar systems may based on, for example insect steroid hormones rather than on mammalian steriod hormones. Indeed, a system based on the ecdysone receptor ofHeliothis virescens has recently been described. Benzene sulphonamide gene switching systems are also known (Hershey et al, Plant Mol. Biol., 17, 679-690 (1991) as are systems based on the alcR protein from Aspergillus nidulans and glutathione S-transferase promoters.

Several genes which confer herbicide resistance are also known, for example, one herbicide to which resistance genes have been described and which is extremely widely used is N-phosphonomethyl-glycine (glyphosate) and its agriculturally acceptable salts including the isopropylamine, trimethylsulphonium, sodium, potassium and ammonium salts.

In a first aspect of the present invention there is provided a chemically inducible plant gene expression cassette comprising an inducible promoter operatively linked to a target gene which confers resistance to a herbicide.

Any herbicide resistance gene may be used but genes which confer resistance to Nphosphonomethyl-glycine or salts or derivatives thereof are especially preferred.

Several inducible promoters may be used to confer the inducible resistance and these include any of those listed above.

__iiij WO 97/06269 PCT/GB96/01883 -3- However, a particularly useful gene switch for use in this area is based on the alc R regulatory protein from Aspergillus nidulans which activates genes expression from the alcA promoter in the presence of certain alcohols and ketones. This system is described in our International Patent Publication No. W093/21334 which is incorporated herein by reference.

The alcA/alcR gene activation system from the fungus Aspergillus nidulans is also well characterised. The ethanol utilisation pathway in A. nidulans is responsible for the degradation of alcohols and aldehydes. Three genes have been shown to be involved in the ethanol utilisation pathway. Genes alcA and alcR have been shown to lie close together on linkage group VII and aldA maps to linkage group VIII (Pateman JH et al, 1984, Proc. Soc.

Lond, B217:243-264; Sealy-Lewis HM and Lockington RA, 1984, Curr. Genet, 8:253-259).

Gene alcA encodes ADHI in A. nidulans and aldA encodes AldDH, the second enzyme responsible for ethanol utilisation. The expression of both alcA and aldA are induced by ethanol and a number of other inducers (Creaser EH et al, 1984, Biochemical J, 255:449-454) via the transcription activator alcR. The alcR gene and a co-inducer are responsible for the expression ofalcA and aldA since a number of mutations and deletions in alcR result in the pleiotropic loss of ADHI and aldDH (Felenbok B et al, 1988, Gene, 73:385-396; Pateman et al, 1984; Sealy-Lewis Lockington, 1984). The ALCR protein activates expression from alcA by binding to three specific sites in the alcA promoter (Kulmberg P et al, 1992, J. Biol.

Chem, 267:21146-21153).

The alcR gene was cloned (Lockington RA et al, 1985, Gene, 33:137-149) and sequenced (Felenbok et al, 1988). The expression of the alcR gene is inducible, autoregulated and subject to glucose repression mediated by the CREA repressor (Bailey C and Arst HN, 1975, Eur. J. Biochem, 51:573-577; Lockington RA et al, 1987, Mol. Microbiology, 1:275- 281; Dowzer CEA and Kelly JM, 1989, Curr. Genet, 15:457-459; Dowzer CEA and Kelly JM, 1991, Mol. Cell. Biol, 11:5701-5709). The ALCR regulatory protein contains 6 cysteines near its N terminus co-ordinated in a zinc binuclear cluster (Kulmberg P et al, 1991, FEBS Letts, 280:11-16). This cluster is related to highly conserved DNA binding domains found in transcription factors of other ascomycetes. Transcription factors GAL4 and LAC9 have been shown to have binuclear complexes which have a cloverleaf type structure containing two Zn(II) atoms (Pan T and Coleman JE, 1990, Biochemistry, 29:3023-3029; Halvorsen YDC et al, 1990, J. Biol. Chem, 265:13283-13289). The structure of ALCR is WO 97/06269 PCT/GB96/01883 -4similar to this type except for the presence of an asymmetrical loop of 16 residues between Cys-3 and Cys-4. ALCR positively activates expression of itself by binding to two specific sites in its promoter region (Kulmberg P et al, 1992, Molec. Cell. Biol, 12:1932-1939).

The regulation of the three genes, alcR, alcA and aldA, involved in the ethanol utilisation pathway is at the level of transcription (Lockington et al, 1987; Gwynne D et al, 1987, Gene, 51:205-216; Pickett et al, 1987, Gene, 51:217-226).

There are two other alcohol dehydrogenases present in A. nidulans. ADHII is present in mycelia grown in non-induced media and is repressible by the presence of ethanol. ADHII is encoded by alcB and is also under the control ofalcR (Sealy-Lewis Lockington, 1984).

A third alcohol dehydrogenase has also been cloned by complementation with a adh- strain of S. cerevisiae. This gene alcC, maps to linkage group VII but is unlinked to alcA and alcR.

The gene, alcC, encodes ADHIII and utilises ethanol extremely weakly (McKnight GL et al, 1985, EMBO J, 4:2094-2099). ADHIII has been shown to be involved in the survival ofA.

nidulans during periods of anaerobic stress. The expression ofalcC is not repressed by the presence of glucose, suggesting that it may not be under the control ofalcR (Roland LJ and Stromer JN, 1986, Mol. Cell. Biol, 6:3368-3372).

In summary, A. nidulans expresses the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when it is grown in the presence of various alcohols and ketones. The induction is relayed through a regulator protein encoded by the alcR gene and constitutively expressed. In the presence of inducer (alcohol or ketone), the regulator protein activates the expression of the alcA gene. The regulator protein also stimulates expression of itself in the presence of inducer. This means that high levels of the ADHI enzyme are produced under inducing conditions when alcohol or ketone are present). Conversely, the alcA gene and its product, ADH are not expressed in the absence of inducer.

Expression of alcA and production of the enzyme is also repressed in the presence of glucose.

Thus the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer by the protein/alcohol or protein/ketone combination).

The alcR and alcA genes (including the respective promoters) have been cloned and sequenced (Lockington RA et al, 1985, Gene, 33:137-149; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).

WO 97/06269 PCT/GB96/01883 Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region ofadh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. However, no equivalent to the alcR regulator protein has been found in any plant. Hence the alcR/alcA type of gene regulator system is not known in plants.

Constitutive expression of alcR in plant cells does not result in the activation of endogenous adh activity.

According to a second aspect of the invention, there is provided a chemically-inducible plant gene expression cassette comprising a first promoter operatively linked to an alcR regulator sequence which encodes an alcR regulator protein, and an inducible promoter operatively linked to a target gene which confers herbicide resistance, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.

The inducible promoter is preferably derived from the alcA gene promoter but may, alternatively be derived from alcR, aldA or other alcR-induced genes.

We have found that the alcA lalcR switch is particularly suited to drive herbicide tolerance genes for at least the following reasons.

I. The alcA/alcR switch has been developed to drive high levels of gene expression. In addition, the regulatory protein alcR is preferably driven from a strong constitutive promoter such as polyubiquitin. High levels of induced transgene expression, comparable to that from a strong constitutive promoter, such as 35 CaMV, can be achieved.

2. If a gene switch is to be used in a situation where the activating chemical is applied simultaneously with the herbicide, a rapid elevation in the levels of herbicide resistance gene is required. Figure 1 reveals a time course of marker gene expression (CAT) following application of inducing chemical. This study shows a rapid increase (2 hours) of CAT expression following foliar application of inducing chemical. The immediate early kinetics of induction are brought about be expressing the regulatory protein in constitutive manner, therefore no time lag is encountered while synthesis of transcription factors takes place. In addition we have chosen a simple two component system which does not rely on a complex signal transduction system.

WO 97/06269 PCT/GB96/01883 -6- 3. We have tested the specificity ofalcA/alcR system with a range of solvents used in agronomic practice. A hydroponic seedling system revealed that ethanol, butan-2-ol and cyclohexanone all gave high levels of induced reporter gene expression (Figure 2).

In contrast when the alcohols and ketones listed in Table 1 in which are used in agronomic practice were applied as a foliar spray only ethanol gave high levels of induced reporter gene activity (Figure 3).

Table 1 1. Isobutyl methyl ketone 13. acetonyl acetone 2. Fenchone 14. JF5969 (cyclohexanone) 3. 2-heptanone 15. N-methyl pyrrolidone 4. Di-isobutyl ketone 16. polyethylene glycol 5-methyl-2-hexanone 17. propylene glycol 6. 5-methylpentan-2,4-diol 18. acetophenone 7. ethyl methyl ketone 19. JF4400 (methylcyclohexanone) 8. 2-pentanone 20. propan-2-ol 9. glycerol 21. butan-2-ol y-butyrolactone 22. acetone 11. diacetone alcohol 23. ethanol 12. tetrahydrofurfuryl alcohol 24. This is of significance since illegitimate induction oftransgenes will not be encountered by chance exposure to formulation solvents. Ethanol is not a common component of agrochemical formulations and therefore with appropriate spray management can be considered as a specific inducer of the alc A R gene switch in a field situation.

4. A range of biotic and abiotic stresses for example pathogen infection, heat, cold, drought, wounding, flooding have all failed to induce the alcA lalcR switch. In addition a range of non-solvent chemical treatments for example salicylic acid, ethylene, absisic acid, auxin, gibberelic acid, various agrochemicals, all failed to induce the alcA alcR system.

WO 97/06269 PCT/GB96/01883 -7- The first promoter may be constitutive or tissue-specific, developmentallyprogrammed or even inducible. The regulator sequence, the alcR gene, is obtainable from Aspergillus nidulans, and encodes the alcR regulator protein.

The inducible promoter is preferably the alcA gene promoter obtainable from Aspergillus nidulans or a "chimeric" promoter derived from the regulatory sequences of the alcA promoter and the core promoter region from a gene promoter which operates in plant cells (including any plant gene promoter). The alcA promoter or a related "chimeric" promoter is activated by the alcR regulator protein when an alcohol or ketone inducer is applied.

The inducible promoter may also be derived from the aldA gene promoter, the alcB gene promoter or the alcC gene promoter obtainable from Aspergillus nidulans.

The inducer may be any effective chemical (such as an alcohol or ketone). Suitable chemicals for use with an alcA/alcR-derived cassette include those listed by Creaser et al (1984, Biochem J, 225, 449-454) such as butan-2-one (ethyl methyl ketone), cylcohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, ethanol.

The gene expression cassette is responsive to an applied exogenous chemical inducer enabling external activation of expression of the target gene regulated by the cassette. The expression cassette is highly regulated and suitable for general use in plants.

The two parts of the expression cassette may be on the same construct or on separate constructs. The first part comprises the regulator cDNA or gene sequence subcloned into an expression vector with a plant-operative promoter driving its expression. The second part comprises at least part of an inducible promoter which controls expression of a downstream target gene. In the presence of a suitable inducer, the regulator protein produced by the first part of the cassette will activate the expression of the target gene by stimulating the inducible promoter in the second part of the cassette.

In practice the construct or constructs comprising the expression cassette of the invention will be inserted into a plant by transformation. Expression of target genes in the construct, being under control of the chemically switchable promoter of the invention, may then be activated by the application of a chemical inducer to the plant.

Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti _i;;n WO 97/06269 PCT/GB96/0883 -8plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation and pollen tube transformation. The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.

Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.

The invention further provides a plant cell containing a gene expression cassette according to the invention. The gene expression cassette may be stably incorporated in the plant's genome by transformation. The invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.

The invention further provides a method for controlling plant gene expression comprising transforming a plant cell with a chemically-inducible plant gene expression cassette which has a first promoter operatively linked to an alcR regulator sequence which encodes an alcA regulator protein, and an inducible promoter operatively linked to a target gene which confers herbicide resistance, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.

This strategy of inducible expression of herbicide resistance can be achieved with a pre-spray of chemical activator or in the case of slow acting herbicides, for example Nphosphonomethyl-glycine (commonly known as glyphosate), the chemical inducer can be added as a tank mix simultaneously with the herbicide.

This strategy can be adopted for any resistance conferring gene/corresponding herbicide combination. For example, the alcA/alcR gene switch can be used with: 1. Maize glutathione S-transferase (GST-27) gene (see our International Patent Publication No W090/08826), which confers resistance to chloroacetanilide herbicides such as acetochlor, metolachlor and alachlor.

2. Phosphinotricin acetyl transferase (PAT), which confers resistance to the herbicide commonly known as glufosinate.

WO 97/06269 PCT/GB96/01883 -9- 3. Acetolactate synthase gene mutants from maize (see our International Patent Publication No W090/14000) and other genes, which confer resistance to sulphonyl urea and imadazlonones.

4. Genes which confer resistance to glyphosate. Such genes include the glyphosate oxidoreductase gene (GOX) (see International Patent Publication No. W092/00377 in the name of Monsanto Company); genes which encode for 5-enolpyruvyl-3phosphoshikimic acid synthase (EPSPS), including Class I and Class II EPSPS, genes which encode for mutant EPSPS, and genes which encode for EPSPS fusion peptides such as that comprised of a chloroplast transit peptide and EPSPS (see for example EP 218 571, EP 293 358, W091/04323, W092/04449 and W092/06201 in the name of Monsanto Company); and genes which are involved in the expression of CPLyase, Various further preferred features and embodiments of the present invention will now be described in the non-limiting examples set out below and with reference to the drawings in which: Figure 1 illustrates the time course of marker gene expression (CAT) following application of inducing chemical; Figure 2 illustrates the levels of induced reporter gene expression on root drenching with a range of solvents; Figure 3 illustrates the levels of induced reporter gene activity when the chemicals listed in Table 1 were applied as a foliar spray; Figure 4 illustrates the production of the 35S regulator construct by ligation ofalcR cDNA into pJR1.

Figure 5 illustrates the production of the reporter construct; Figure 6 is a summary of the cassettes and specific plant transformation constructs; Figure 7 illustrates the chloroplast transit sequence 1 from Arabidopsis RUBISCO (CPT 1); Figure 8 shows the sequence of plasmid pMJB 1; Figure 9 is a map ofplasmid pJRIi; Figure 10 illustrates the chloroplast transit sequence CTP2 from EPSPS class I gene from Petunia hybrida; Figure 11 is a map of plasmid pUB-1;

I

9a- Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

Various further preferred features and embodiments of the present invention will now be described in the non-limiting examples set out below and with reference to the drawings in which: Figure 1 illustrates the time course of marker gene expression (CAT) following application of inducing chemical; Figure 2 illustrates the levels of induced reporter gene expression on root drenching with a range of solvents; Figure 3 illustrates the levels of induced reporter gene activity when the chemicals listed in Table 1 were applied as a foliar spray; S* Figure 4 illustrates the production of the 35S regulator construct by ligation of alcR cDNA into pJR1.

15 Figure 5 illustrates the production of the reporter construct; Figure 6 is a summary of the cassettes and specific plant transformation constructs; Figure 7 illustrates the chloroplast transit sequence 1 from Arabidopsis RUBISCO (CPT 1); Figure 8 shows the sequence of plasmid pMJB1; Figure 9 is a map of plasmid pJR1i; Figure 10 illustrates the chloroplast transit sequence CTP2 from EPSPS class 1 gene from Petunia hybrida; Figure 11 is a map of plasmid pUB-1; li:i-il~Y--LT~~ i-l-- WO 97/06269 PCT/GB96/01883 Figure 12 is a map ofplasmid pMF6; Figure 13 is a map of plasmid pIE109 in which the numbers are in base pairs (not to scale) and the following abbreviations are used: ADHi Alcohol dehydrogenase from maize; PAT Phosphinothricin acetyl transferase (Basta resistance gene); AMP Ampicillin resistance gene; CaMV 35S Cauliflower mosaic virus 35S promoter; nos Poly A Nopaline synthase poly A region; ori ColE 1 origin of replication from pUC Figure 14 is a map of plasmid pMVI in which the numbers are in base pairs (not drawn to scale) and the abbreviations are as for Figure 13 with the following additional abbreviations: UBQp Maize ubiquitin promoter; UBQi Maize ubiquitin intron; nos Nopaline synthase 3' terminator; CZP GOX Chloroplast transit peptide glyphosate oxidase sequence; CZP2 GPSPS Chloroplast transit peptide EPSP synthetase sequence; Figure 15 shows the preparationof plasmid pUC4 by ligation of pAr3 and pBSSK; Figure 16 is a map ofplasmid pMV2 in which the numbers are in base pairs (not drawn to scale) and the abbreviations are as for Figure 14 with the following additional abbreviations: AlcA Aspergillus nidulans alcA promoter; AlcR Aspergillus nidulans alcR promoter; Figure 17 is a map of plasmid pDV1-pUC; Figure 18 is a map of plasmid pDV2-pUC; Figure 19 is a map ofplasmid pDV3-Bin; Figure 20 is a map of plasmid pDV4-Bin; and Figure 21 is a western Blot showing the expression of EPSPS and GOX in transformants.

WO 97/06269 PCT/GB96/01883 -11-

EXAMPLES

We have chosen to exemplify the alcA/alcR gene switch with genes conferring resistance to glyphosate. The switch will be used to drive inducible expression of glyphosate oxidase (GOX) in plants. Switchable GOX has been expressed alone or in conjunction with constitutive expression of 5-enol-pyruvylshikimate 3-phosphate (EPSPS) CP4. Constructs have been optimised for expression in monocotyledonous and dicotyledonous crop species.

EXAMPLE 1 Production Of The alcR Regulator Construct.

The alcR genomic DNA sequence has been published, enabling isolation of a sample of acR cDNA.

The alcR cDNA was cloned into the expression vectors pJRI(pUC). pJR contains the Cauliflower Mosaic Virus 35S promoter. This promoter is a constitutive plant promoter and will continually express the regulator protein. The nos polyadenylation signal is in the expression vector.

Figure 4 illustrates the production of the 35S regulator construct by ligation ofalcR cDNA into pJR1. Partial restriction of the alcR cDNA clone with BamHI was followed by electrophoresis in an agarose gel and the excision and purification of a 2.6 Kb fragment. The fragment was then ligated into the pJR1 vector which had been restricted with BamHI and phosphatased to prevent recircularisation. The alcR gene was thus placed under control of the CaMV 35S promoter and the nos 3' polyadenylation signal in this "35S-alcR" construct.

EXAMPLE 2 Production Of The alcA-CAT Reporter Construct Containing The Chimeric Promoter.

The plasmid pCaMVCN contains the bacterial chloramphenicol transferase (CAT) reporter gene between the 35S promoter and the nos transcription terminator (the "35S-CAT" construct).

The alcA promoter was subcloned into the vector pCaMVCN to produce an "alcA-CAT" construct. Fusion of part of the alcA promoter and part of the 35S promoter created a chimeric promoter which allows expression of genes under its control.

Figure 5 illustrates the production of the reporter construct. The alcA promoter and the 35S promoter have identical TATA boxes which were used to link the two promoters together using a recombinant PCR technique: a 246 bp region from the alcA promoter and WO 97/06269 PCT/GB96/01883 -12the 5' end of the CAT gene from pCaMVCN (containing part of the -70 core region of the promoter) were separately amplified and then spliced together using PCR. The recombinant fragment was then restriction digested with BamHI and HindIII. The pCaMVCN vector was partially digested with BamHI and HindIII, then electrophoresed so that the correct fragment could be isolated and ligated to the recombinant fragment.

The ligation mixtures were transformed into E coli and plated onto rich agar media.

Plasmid DNA was isolated by miniprep from the resultant colonies and recombinant clones were recovered by size electrophoresis and restriction mapping. The ligation junctions were sequenced to check that the correct recombinants had been recovered.

EXAMPLE 3 Glyphosate Resistance Constructs A summary of the cassettes and specific plant transformation constructs is shown in Figure 6.

Dicot Vector 1 Vector 1 is a constitutive control plasmid containing the glyphosate oxidase gene (GOX) fused to the chloroplast transit sequence 1 from Arabidopsis RUBISCO (CPT 1) (Figure 7) driven by the enhanced 35S CaMV promoter (ES) and the TMV omega translational enhancer sequence (TMV). Vector 1 utilizes the nopoline synthase terminator (nos). The synthetic GOX gene with the addition of CTP 1 was synthesised with information from patent publication W092/00377 with addition of NcoI site at the translation start ATG, and a Kpn I at the 5' end. Internal Sph I sites and NcoI site were deleted during synthesis with no change in amino acid usage. The CTP 1 GOX synthesised sequence was isolated as a Nco I Kpn I fragment and ligated using standard molecular cloning techniques into NcoI KpnI cut pMJB 1, a plasmid based on pIBT 211 containing the CaMV 35 promoter with duplicated enhancer linked to the tobacco mosaic virus translational enhancer sequence replacing the tobacco etch virus 5' non-translated leader, and terminated with the nopaline synthase poly signal (nos) (Figure 8).

A cassette containing enhanced 35 CaMV TMV sequence CTP 1 GOX and nos terminator (dicot vector 1 pUC Figure 17) was isolated as a HindIII EcoRI fragment and ligated into Hind III EcoRI cut pJRIi, a Bin 19 base plant transformation vector (Figure 9).

I~ i i WO 97/06269 PCT/GB96/01883 13 Dicot Vector 2 The synthetic EPSPS CP4 gene, fused to the chloroplast transit sequence CTP2 (Figure 10) from EPSPS class I gene from Petunia hybrida, was synthesised with data from patent WO 92/04449 with NcoI at the translation initiation ATG. A internal Sph I site was silenced in the EPSPS CP4 gene with no change of amino acid usage.

A fragment containing the synthetic CTP 2 CP4 EPSPS was isolated as a Ncol Sac I fragment and ligated in to pMJBI. A fragment containing the CaMV 35 promoter with a duplicated enhancer, TMV omega sequence CTP 2 transit peptide, EPSPS and nos terminator was isolated as a EcoRI Hind III fragment (dicot vector 2 pUC Figure 18)and cloned into pJRIi to give dicot vector 2 pUC (Figure 18).

Upon sequencing the junctions of dicot vector 2, an additional sequence was identified inserted between the SacI site and the beginning of the nos terminator. This was as follows: AGG CTG CTT GAT GAG CTC GGT ACC CGG GGA TCC ATG GAG CCG AAT 3' Dicot Vector 3 A control vector with both EPSPS and GOX genes was constructed by cutting dicot vector 2 with EcoRI and inserting an AEcoRI Sph I AEcoRI linker. The sequence of the linker is shown below: 5' AAT TAG GGG CAT GCC CCT 3' The resultant vector was cut with Sph I to liberate the cassette B which was cloned into an SphI site in dicot vector 5' to the 35 CaMV promoter. Cassettes 1) and 2) were then excised as a HindIII and EcoRI fragment from dicot vector 3- pUC (Figure 19) and cloned in to pJRIi.

Dicot Vector 4 An inducible GOX vector was constructed by excising the CAT gene from "p alcCAT" as PstI fragment. The vector band, containing the alcA promoter and nos terminator was gel purified and used in ligations with a PstI-XhoI-KpnI-PstI linker, the sequence of which is as follows: 5' GCC ACT CGA GCT AGG TAC CCT GCA 3' The orientation of this was confirmed by sequence analysis. The TMV omega and CTPI GOX sequence from dicot vector 1) were isolated as a XhoI KpnI fragment and cloned into the alcA nos vector containing the XhoI-KpnI-PstI linker. The alcA TMV CTP 1 GOX WO 97/06269 PCT/GB96/01883 14nos cassette was excised as a IHindIII fragment and cloned into the plant transformation vector "p35S-alc containing the alcR cDNA nos terminator under the control of the CaMV promoter to form dicot vector 4 (Figure Dicot Vector Dicot vector 5 (Figure 22) containing inducible GOX and constitutive EPSPS genes was prepared using the following cloning strategy. Dicot vector 2 (pDV2 -pUC) was modified by cloning in a AEcoRI-IindIII-AEcoRI linker into the EcoRI site to allow excision of the CaMV en-CTP2-EPSPS -nos cassette as a HindIII fragment. This fragment was then ligated into HindlII cut pDV4-Bin. Recombinants containing all three cassettes ie 1o CaMVen-CTP2-EPSPS-nos and AlcA-CTP1-GOX-nos were selected by hybridization with radiolabelled oligonucleotides. Confirmation of orientation was done by sequencing across all borders.

Monocot Vectors Vector 1: Cassette D An EcoRI-Notl-EcoRI linker (5'AATTCATTTGCGGCCGCAAATG3') was inserted into dicot vector pDV1. The plasmid was cut with NcoI and the 5' overhang filled-in with DNA Polymerase I Klenow fragment. The linear vector was then cut with NotI and the resulting blunt/NotI fragment containing the CTP1 GOX and nos terminator was ligated into a Smal/NotI digested pPUB1 vector (Figure 12) containing the polyubiquitin promoter, polyubiquitin intron with a KpnI-Notl-KpnI linker AAATGGTAC3') insertion. A HindlII-Notl-HindIII linker CGCTGCA3') was inserted into the resulting construct.

Vector 1: Cassette E An EcoRI-NotI-EcoRI linker (5'AATTCATTTGCGGCCGCAAATG3') was inserted into dicot vector pDV2. The plasmid was cut with Ncol and the 5' overhang filled-in with DNA Polymerase I Klenow fragment. The linear vector was then cut with NotI and the resulting blunt/NotI fragment containing the CTP2 EPSPS and nos terminator was ligated into a SnaI/NotI digested pPUB I vector containing the polyubiquitin promoter, polyubiquitin intron with a KpnI-NotI-KpnI linker (5'CATTTGCGGCCGCAAATGGT AC3') insertion to create plasmid 1. The PAT selectable marker cassette (35S CaMV promoter, Adhl intron, =-iii t WO 97/06269 PCT/GB96/01883 phosphinothricin acetyl transferase gene (PAT), nos terminator) was excised from pIE108 (Figure 14) and cloned into the HindIII site on plasmid 1 to give mononcot cassette E.

Diagnostic restriction digestion was used to confirm that the selectable marker cassette was inserted 5' to 3' in the same orientation as the CTP2 EPSPS cassette.

A fragment containing the polyubiquitin promoter, polyubiquitin intron, CTP 1 GOX and nos terminator was excised from cassette D with NotI and ligated into NotI cassette E to form monocot vector 1 (Figure 14). Restriction digestion was used to confirm that the two cassettes were inserted in the same orientation.

The selectable marker cassette (35 CaMV promoter, Adhl intron, phosphinothricin acetyl transferase gene (PAT), nos) was excised from pIE108 and cloned into the Hind III site in 5) to give monocot cassette E.

Vector 1 A fragment containing the polyubiquitin promoter, polyubiquitin intron GOX and nos was exised from cassette D with NoIl and cloned into NotI cut casette E, to form monocot vector 1.

Vector 2 Cassette F An EcoRI fragment from pUC4 (Figure 15) containing the alcR cDNA and nos terminator sequences was blunt end-filled with DNA Polymerase I Klenow fragment, ligated into pUBI with the KpnI-NotI-KpnI linker insertion and orientated by restriction analysis. The PAT selectable marker cassette was inserted in the HindIII site after excision from pIE108 and orientated by restriction analysis to create vector 1. Plasmid 1 above containing the polyubiquitin promoter, polyubiquitin intron, CTP2 EPSPS and nos terminator was cut with HindIII and a AHindIII-NotI-HindIII linker: 5'AGCTCGCAGCGGCCGCTGCA3' 5'GCGTCGCCGGCGACGTTCGA3' inserted and orientated by sequencing to create vector 2.

A ClaI-NcoI-ClaI linker (5'CGATGCAGCCATGGCTGCAT3') was inserted into pMF6 (Figure 13) to give vector 3. An Ncol/KpnI fragment containing CTP1 GOX was excised from pDV1 and inserted into Ncol/KpnI cut vector 3 to create vector 4. A Sall fragment containing the maize AdhI intron, CTP1 GOX was excised from vector 4 and ligated into Sall cut pUC2 containing the alcA promoter and nos terminator and orientated by sequencing to create vector 5. A HindII fragment from vector 5 containing the alcA promoter, maize Adhl :i WO 97/06269 PCT/GB96/01883 16intron, CTP GOX and nos terminator was ligated into IindIII cut vector 2 and orientated by restriction digestion. A NotI fragment from the resulting construct containing polyubiquitin promoter, polyubiquitin intron, CTP2 EPSPS, nos terminator, alcA promoter, maize AdhI intron, CTP1 GOX and nos terminator was ligated into NotI cut vector 1 and orientated by restriction analysis to create monocot vector 2 (Figure 16).

EXAMPLE 4 Plant Transformation Plasmids for dicot transformation were transferred to Agrobacterium tumefaciens LBA4404 using the freeze thaw method described by Holsters et al 1978.

Tobacco transformants were produced by the leaf disc method described by Bevan 1984. Shoots were regenerated on a medium containing 100 mg/l kanamycin. After rooting plants were transferred to the glasshouse and grown under 16h light/8h dark conditions.

Oilseed rape (Brassica napus cv westar) transformations were performed using the cotyledon petiole method described by Moloney et al 1989. Selection of transformed material was performed on kanamycin (15 mg/1). Rooted shoots were transferred directly to a soil based compost and grown to maturity under controlled glasshouse conditions (16h day 0 C day, 15 0 C night 60% RH).

Maize transformation was performed using the particle bombardment approach as described by Klein et al 1988. Selections were performed on 1 mg/1 biolophos.

Sugar beet transformation was performed using the guard cell protoplast procedure see our International Patent Publication No. W095/10178.

Results showing details of the transgenic plants obtained are shown in Tables 2 and 3 below.

Table 2 Transformation Details For Tobacco Vector Species Shoots removed Rooted pDVI Tobacco 150 57 pDV2 Tobacco 150 pDV3 Tobacco 270 77 pDV4 Tobacco 350 135 Tobacco 150 WO 97/06269 PCT/GB96/01883 17- Table 3 Transformation Details in Oil Seed Rape Vector Species Shooting Calli Rooted pDVI OSR 14 shoots from 14 pDV2 OSR 13 shoots from 13 pDV3 OSR 18 shoots from 18 pDV4 OSR 20 shoots from pDVS OSR 19 shoots from 18 EXAMPLE t0 Transgenic Plant Analysis Polymerase Chain Reaction (PCR) Genomic DNA for PCR analysis of transgenic plants was prepared according to the method described by Edwards et al 1992. PCR was performed using conditions described by Jepson et al Plant Molecular Biology Reporter, 13 1-13 8 (199 Primer sets were designed for each of the introduced cassettes.

The plants were analysed using the following oligonucleotide combinations:pDVI TMV1 GOXI, GOX3 nos I pDV2 TMVI +EPSPS 1, EPSPS3 +noslI pDV3 EPSPS3 +GOX1 pDV4 35S +AIcR1, AlcA2 +GOX1I 35S AcRI, AlcA2 +GOXLI, TMV I +EPSPS1I Oligonucleotide sequences are given below:- TMVI 5' CTCGAGTATTTTTACAACAATTACCAAC GOXi 5' AATCAAGGTAACCTTGAATCCA GOX3 5' ACCACCAACGGTGTTCTTGCTGTTGA NOS 1 5' GCATTACATGTTAATTATTACATGCTT EPS PS 1 5' GTGATACGAGTTTCACCGCTAGCGAGAC EPSPS3 5' TACCTTGCGTGGACCAAAGACTCC 35s 5' GTCAACATGGTGGAGCACG AlcR1 5' GTGAGAGTTTATGACTGGAGGCGCATC AlcA2 5' GTCCGCACGGAGAGCCACAAACGA WO 97/06269 PCT/GB96/01883 18- Selection on Glvphosate Kill Curves for Tobacco var Samsun and Brassica napus var Westar on glyphosate Both species were tested on a range of glyphosate concentrations by inserting, in the case of tobacco a 5-6mm stem segment carrying a leaf node and in the case of oil seed rape the growing tip plus two leaves into MS medium containing glyphosate at 0, 0.0055, 0.011, 0.0275, 0.055 and 0.01 mM glyphosate isopropylamine salt. The results were scored after two weeks growth as and are given in Table 4 below.

Table 4 Conc Westar Tobacco 0 Good stem growth, 4-5 new leaves, roots up to 5cm As OSR 0.005 No stem growth, 1 new leaf, roots to Icm No growth in any organ 0.011 No stem growth, no new leaves, 0.0275 No stem growth, no new leaves, roots-2mm 0.055 No growth in any organ, ends of stem blackened 0.01 As for 0.055mM Selection for glyphosate tolerant transformants was performed on glyphosate concentrations of 0.01 and 0.05mM.

Constitutively tolerant plants Following from the data obtained on wild type plants, pDV1,2 and 3 PCR +ve primary transformants were screened on MS medium containing glyphosate at the levels described above. For tobacco this was done by inserting three or four stem sections per transformant into the medium and using untransformed Samsun as control. Scoring was based on the behaviour of the majority. Plants showing tolerance at the higher concentration of herbicide were grown on to maturity in the glass house, for seed collection.

Segregation Test Seeds were sterilized in 10% bleach for 10 min. After several washes in sterile water 200 seeds were sown on 1/2 MS medium (2.3 g/l MS salt, 1.5% sucrose, 0.8% Bactoagar, i_

I:

WO 97/06269 PCT/GB96/01883 -19pH 5.9) containing 100 mg/l kanamycin. Seeds were grown at 26 0 C with 16 hours/8 hours light/dark prior to scoring.

Western Analysis Antibody Generation GOX and EPSPS protein were over expressed in E.Coli using a pET expression system. Following IPTG induction GOX and EPSPS were electro eluted from the shake flask grown cell paste and used to immunise rabbits (two animals per clone).

Preparation of Tissue Extracts for Immunoblotting 120 mg of leaf tissue plus 60 mg PVPP and 500 tl extraction buffer (50 mM Tris-HCI pH 8, 1 mM EDTA, 0.3 mM DTT) were ground with a blender for several minutes. After homogenisation the extract was centrifuged at 15,000 rpm for 15 min. The supernatant was stored at -80° C until required. Protein concentrations in the extract were measured according to Bradford.

SDS-PAGE and Immunoblotting 25 pg protein were separated by SDS-PAGE. The running buffer was 14.4 (w/v) glycine, 1 SDS and 3 Tris Base. The samples were loaded according to Laemmli.

After SDS-PAGE proteins were electroblotted overnight with 40 mA onto nitrocellulose (Hybond

T

C, Amersham) using an electroblot unit from Biorad. The membrane was stained in 0.05 CPTS dissolved in 12 mM HC1. Blots were rinsed in 12 mM HCI and destained for 5 min in 0.5 M NaHCO3 followed by an intensive rinse with H 2 0. Membranes were then blocked, immunodetected and washed according to the Amersham ECL kit. Indirect immunodetections were performed with a 1:10000 dilution of a rabbit anti-GOX or anti-EPSPS polyclonal as first antibody and with a 1:1000 dilution of an anti-rabbit second antibody, associated with horseradish peroxidase. An additional wash was carried out overnight to eliminate background. Detection was performed using the ECL kit from Amersham and the results are shown in Figure 21 in which Lane is the control and the remaining lanes are transformants. The western analysis demonstrates that some transformants are capable of expressing GOX and EPSPS.

Constitutively tolerant plants Cell extracts were prepared from each glyphosate tolerant plant and the amount of expresssed protein estimated by western analysis using antibody appropriate to the WO 97/06269 PCT/GB96/01883 transformant. Plants expressing very high levels of GOX or EPSPS were tested on higher levels of glyphosate to relate level of expression to herbicide tolerance.

Inducibly tolerant plants To demonstrate inducible tolerance to glyphosate PCR positive primary transformants from the transformations with pDV4 and 5 were transferred directly to the glass house. After two weeks the plants were induced by an ethanol root drench solution) and left for 24 hours prior to western analysis performed to assess level of expression of GOX after induction. After a period of time to allow the plants to return to the uninduced state, the western analysis was repeated to allow selection ofinducibly tolerant plants. Plants which showed the highest levels of GOX expression following ethanol treatment were taken forward to time course analysis. GOX levels were assessed at 6, 12, 18, 24, 36, 48 hours following ethanol treatment, by western analysis.

High expressing GOX plants for both pDV4 and pDV5 were used in glass house trials to demonstrate inducible glyphosate tolerance. Plants were induced using a range of ethanol concentrations by root drench application to pot grown plants. Following GOX induction plants were sprayed with glyphosate. Wild type controls and uninduced plants were also treated with herbicide.

Northern Analysis Primary transformants containing dicots vector and were analysed by northern blot analysis using a CTP2 EPSPS probe as a Ncol Sac I fragment. Primary transformants containing the dicot vectors were analysed by northern blotting using a CTPI GOX probe as a Ncol KpnI fragment. Similarly, transgenic corn lines containing monocot vectors and were analysed using a CTP2 EPSPS probe.

Transformants containing dicot vector or monocot vector were treated with a foliar application of 5% ethanol to induce GOX levels. RNA was isolated 24 hours after treatment and subjected to northern analysis with a CTPI GOX probe.

Primary transformants which were PCR positive for the appropriate cassettes and showed GOX or EPSPS transcript levels were taken for further analysis.

Glyphosate Oxidoreductase Assay Assays for glyphosate oxidoreductase were carried out as described by Kishore and Barry (WO 92/00377). These entailed measuring glyphosate dependent uptake of oxygen WO 97/06269 PCT/GB96/01883 -21 using an oxygen electrode, detection ofglyoxylate formation by reaction with 2, 4 dinitrophenylhydrazine and determination of the hydrozone using HPLC or, preferably, using 14 C] glyphosate as the substrate and detecting the formation of radioactive aminomethyl phosphonic acid via HPLC on an anion exchange column.

EPSPS Assay Assays for 5-enol-pyruvylshikimate-3 -phosphate (EPSP) synthase activity in plant extracts were carried out by following the disappearance of the phosphoenol pyruvate substrate (as described by Rubin, Gaines, C.G and Jensen, in Plant Physiol (1984 839-845) or by conducting the assay in the reverse direction and coupling to pyruvate kinase and lactate dehydrogenase (as described by Mousdale D.M. and Coggins J.R. in Planta (1984) 160, 78-83) or by using 14(-labelled phosphoenol pyruvate as substrate and detecting the formation of radioactive EPSP by HPLC on an anion exchange column and detecting using a flow-through radioactivity detector as described by Della-Cioppa et al in Proc. Nat. Acad. Sci. (USA) (1986), 83, 6873-6877. The latter assay was used to confirm that the EPSP synthase activity was, as expected, relatively resistant to inhibition by glyphosate.

Claims

1. A chemically inducible plant gene expression cassette comprising an inducible promoter which is derived from the alcA, alcR, aldA or other alcR-induced gene promoter operatively linked to a glyphosate oxidase gene which confers resistance to the herbicide N-phosphonomethyl-glycine or a salt or derivative thereof.

2. A chemically inducible gene expression cassette comprising a first promoter operatively linked to an alcR regulator sequence which encodes an alcR regulator protein, and an inducible promoter operatively linked to a glyphosate oxidase gene which confers herbicide resistance, the inducible promoter being :00* activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the glyphosate oxidase gene.

3. A plant gene expression cassette accordingto claim 2, wherein the inducible promoter is derived from the alcA, alcR, aldA or other alcR-induced gene promoter. 0

4. A plant gene expression cassette according to claim 3 wherein the inducible promoter is a chimeric promoter. 0

5. A gene expression cassette comprising a first promoter operably linked to a first target gene which codes for 5-enol- pyruvylshikimate 3-phosphate (EPSPS) CP4 which confers resistance to N-phosphonomethyl-glycine; and a second promoter operably linked to a second target gene coding for glyphosate oxidase which metabolises N-phosphonomethyl-glycine; wherein the second promoter is inducible by the external application of an effective exogenous inducer and is derived from the alcA, alcR, aldA or other alcR-induced gene promoter, and wherein resistance of a plant to N-phosphonomethyl-glycine is provided by expression of the first and/or second target genes. 23

6. The gene expression cassette according to claim 5 wherein the first promoter is a constitutive promoter.

7. A plant cell containing a plant expression cassette according to any one of the preceding claims.

8. A plant cell according to claim 7, wherein the plant gene expression cassette is stably incorporated in the plant's genome.

9. A plant tissue comprising a plant cell according to claim 7 or claim 8. A plant comprising the plant cell according to claim 7 or claim 8. 0

11. A plant derived from the plant according to claim

12. A seed derived from the plant according to claim 10 or claim 1 1.

13. A method of controlling herbicide resistance comprising transforming a plant cell with the plant gene expression cassette according to any one of claims 1 to S"6.

14. A method of selectively controlling weeds in a field of plants according to claim 10 or claim 11, comprising applying an effective amount of the glyphosate and the exogenous inducer. A method of selectively controlling weeds in a field of plants derived from the seed of claim 12, comprising applying an effective amount of the herbicide N-phosphonomethyl-glycine and the exogenous inducer.

16. A gene expression cassette according to claim 1 with reference to the Examples. DATED: 24 AUGUST 1999 PHILLIPS ORMONDE FITZPATRICK ATTORNEYS FOR: ZENECA LIMITED